The goal of this project is to determine the mechanisms by which the endothelial cell protein C receptor (EPCR) functions. The aims grow out of our observations which follow. EPCR is expressed preferentially on the large vessel endothelium and enhances protein C activation. It is found on the cell surface and in discrete organelles include caveolae and is shed in a highly regulated fashion induced by cytokines and thrombin. Thrombin also up-regulates EPCR mRNA levels in vivo. Blocking EPCR-protein C interactions leads to a dramatic exacerbation of the primate response to E. coli resulting in a elevated cytokine levels, DIC, capillary leak, and thrombosis at sites where little EPCR is expressed. EPCR can also undergo nuclear translocation form the plasma membrane with our without carrying activated protein C as cargo. In cultured 293 cells, EPCR transfection results in an altered expression of a limited number of genes without changing the expression of the vast majority of genes. The aims of the present project are to determine whether lateral mobility of EPCR of EPCR in the membrane is important for EPCR acceleration of protein C activation and the EPCR concentration dependence of protein C activation: to determine the sites on EPCR and protein C required for interaction; to determine the topography of the complex by measuring the distance from the active site of APC to the membrane surface when APC is bound to EPCR vs. directly to phospholipid; and to determine the impact of EPCR promoter mutations identified in thrombotic patients on expression in cultured cells. The structural requirements within EPCR and the cellular mechanisms involved in EPCR mediated alteration of gene expression will be investigated. These studies are designed to improve our understanding of the mechanisms by which EPCR functions, provide insights into how EPCR mutations might influence thrombotic risk, and improve our understanding of the role of EPCR in the pathophysiology of thrombotic disease.